Fibrotic-like collagen matrices as 3D in vitro models for investigating the impact of pathological extracellular matrix on skeletal muscle cell behavior

Abstract

Skeletal muscle fibrosis is a hallmark of muscular dystrophies, aging or severe muscle injuries. It is characterized by the extensive accumulation of extracellular matrix (ECM), primarily composed of type I collagen, which disrupts muscle architecture, impairs contractile function, and compromises muscle regenerative abilities. Despite its clinical relevance, to date, no animal or in vitro models recapitulate the main features of muscle fibrosis. In this study, we designed both healthy and fibrotic 3D matrices to investigate the impact of fibrotic environment on myoblast behavior. Fibrotic matrices were engineered by 3D printing of dense collagen solutions in air, followed by slow gelation to yield non-porous, isotropic hydrogels. Subsequent crosslinking using EDC/NHS chemistry yielded matrices with a Young’s modulus of approximately 50 kPa. These fibrotic matrices successfully recapitulated the main characteristics of muscle fibrosis, including compact structure of collagen bundles and increased stiffness, consequently limiting oxygen and nutrient diffusion. C2C12 myoblasts cultured within the fibrotic matrices exhibited altered behavior compared to those grown in healthy matrices. In the fibrotic environment, myoblasts failed to differentiate into mature myotubes, lacked proper alignment, and showed signs of hypoxia. Additionally, they secreted pro-inflammatory cytokines and were unable to remodel their surrounding ECM. Together, these findings underscore the detrimental effects of a fibrotic environment on skeletal muscle cell behavior. The development of these two distinct 3D in vitro muscle models, one representing healthy muscle and the other fibrotic, offers a valuable platform for investigating the pathophysiology of skeletal muscle fibrosis for testing potential therapeutic strategies.